MicroRNAs (miRNAs) are small non-coding RNAs that mediate post-transcriptional gene regulation across a wide variety of Eukaryotes. Misregulation of these regulators has been implicated in cancer and other human diseases. In addition, miRNAs have been identified in conserved pluripotency and developmental pathways. Thus, understanding what controls how miRNAs are expressed will have broad implications for experimentally directing these pathways and for developing therapeutics. Thousands of miRNAs have been discovered computationally in a variety of organisms and confirmed by high-throughput sequencing experiments, yet little characterization has been done to understand mechanisms that govern their biogenesis and expression. For example, there are multiple instances of miRNA genes arranged in an operon-like cluster throughout the genome. Despite being produced from one transcript, often only one miRNA will be principally expressed and this dominance can change during development, suggesting post-transcriptional regulation. The stepwise nature of the miRNA biogenesis pathway, which includes processing by several identified RNase complexes, export from the nucleus, and loading onto the miRNA induced silencing complex (miRISC), allows for regulation at multiple stages after transcription. There are several examples of individual miRNAs that are post-transcriptionally regulated by sequences in their primary transcript and by endogenous proteins. The goal of this proposal is to characterize clustered miRNAs in Caenorhabditis elegans (C. elegans) and to identify cis and trans factors responsible for the regulation of differential expression of clustered miRNAs. A comprehensive analysis of the literature and public sequencing databases reveals several clustered miRNAs that appear subject to post-transcriptional regulation in C. elegans. In Aim1, Rapid Amplification of cDNA Ends (RACE), Northern Blotting, and RT-PCR will be used to define the pri-miRNA transcripts and temporal expression of several potential clustered miRNAs. RNAi knockdown of processing enzymes will facilitate defining the step of regulation in vivo, while processing will be recapitulated and analyzed in vitro using worm extracts. In Aim 2, sequences and structural features important for the biased expression will be discovered by examining mutant transgenic worms and in vitro secondary structure probing. Finally, in Aim 3, we will identify RNA binding proteins that mediate differential expression of clustered miRNAs through RNA pull down and in vitro processing assays. In addition, RNAi will be used to both confirm proteins identified through in vitro methods and screen for elusive interactors.